Collect the cells in growth medium and transfer to a 15 mL conical tube.

Pellet the cells at room temperature at 1000 rpm for 3 min.

Aspirate off the growth medium, but leave enough to cover the pellet. Flick the tube to break up the pellet. Chill the cells on ice.

Do this step twice: Add 1 mL cold FACS buffer to the cells. Pellet the cells at room temperature at 1000 rpm for 3 min. Aspirate off the buffer, leaving enough to cover the pellet. Flick the tube to break up the pellet.

Paraformaldehyde Fixation

Make 1% Paraformaldehyde Solution

Add 1ml of 1% Paraformaldehyde Solution and incubate for 15 minutes on ice. (Longer incubations compromise the quality of CV’s in DNA histograms.)

• Wash 2X with cold FACS Buffer.

Saponin Permeabilization
• Decant the last wash and resuspend pellet in remaining residual volume.
• Resuspend cells in 1 ml FB-S, added slowly while flicking the sample.
• Incubate at RT for 30 minutes.
• Spin down. Decant. Resuspend cell in the residual volume of FB-S. Should be about 100ul.

Antibody Staining (All staining and washes are to be done in FB-S)
• Incubate cells in 100ul final volume of antibody(ies), diluted to recommended concentration in FB-S, for 30 minutes on ice.
• Wash cells 2X in cold FB-S. Decant Supernatant.
• Resuspend in 500ul of FACS Buffer.
• Analyze on Flow cytometer. (keep covered on ice until analysis; always have NEGATIVE control)